A key subunit of myofibrils, the Z-band, links the sarcomeric contractile units in series and also serves as attachment sites for the intermediate filaments, the costameres and membranes. How this myofibrillar complex is assembled is unknown. The first specific aim is to analyze the assembly of Z-bands in live cells in two avian culture systems: precardiac mesoderm cultures in which the first myofibrils form, and 10-day embryonic cardiomyocyte cultures in which new myofibrils assemble in cells with pre-existing myofibrils. The second specific aim is to test the idea that expression of a mutated Z -band titin fragment, corresponding to a mutation reported to be involved in Hypertrophic Cardiomyopathy, will alter Z-band stability, interfere with the ability of alpha-actinin to cross-link actin filaments, and/or induce myofibril disarray in transfected cardiomyocytes. The third specific aim is to analyze the role of a new Z-band protein, NspI-1, in the transition of Z-bodies to Z-bands of mature myofibrils and in the maintenance of Z-band structure. Nspl-1 may be a potentially important molecule for linking Z-bands, costameres, intermediate filaments, and membranes. The experimental approaches include the use of plasmids encoding members of the group of Green Fluorescent Proteins ligated to sarcomeric proteins and mutated domains for transfections of cardiomyocytes. Images of live cells will be analyzed by optical techniques, such as Fluorescence Recovery after Photobleaching (FRAP), confocal and deconvolution microscopy. These advanced optical methods coupled with molecular and biochemical techniques will be used to test hypotheses about the formation of myofibrils and the effects of mutant Z- band titin fragments on the dynamics of proteins interacting with them. These approaches should yield new insights into the role of the Z-band in basic and pathologic processes in myofibril assembly in cardiomyocytes.